鸡支气管堵塞死鸡病变部位感染细菌及其耐药性分析

doi:10.6048/j.issn.1001-4330.2021.11.021

摘要/Abstract

摘要：

【目的】研究以支气管被黄、白色干酪样物质堵塞为主要病理变化的鸡呼吸系统疾病,为研究支气管堵塞形成的细菌学基础提供依据。【方法】无菌采集不同鸡场典型病死鸡的支气管堵塞部位样本,用常规细菌分离法结合16 sDNA测序进行细菌学分析,并对分离菌进行药敏试验和致病性试验。【结果】11个鸡场采集的18个样本中均分离到了大肠杆菌和粪肠球菌,2个鸡场分离到了奇异变形杆菌,1个鸡场分离到了鲍曼不动杆菌,所有典型发病鸡场均未检测到鸡毒支原体和滑液支原体。K-B法药敏实验显示大肠杆菌主要对头孢噻肟、氯霉素、氨苄西林、四环素、复方新诺明等有较高的耐药性;粪肠球菌主要对头孢他啶、氨曲南、复方新诺明等药物耐药。所有分离菌均无致病作用。【结论】大肠杆菌、粪肠球菌、鲍曼不动杆菌和奇异变形杆菌等可单独或同时存在于支气管堵塞部位,可能是该病理变化形成的主要协同因素,而大肠杆菌和粪肠球菌存在于所有的被检病例中,且耐药性普遍很强。

关键词: 鸡支气管堵塞; 大肠杆菌; 粪肠球菌

Abstract:

【Objective】 To study chickens with bronchial obstruction by yellow and white caseous substances. The purpose of this research is to study the bacteriological basis of bronchial obstruction. 【Methods】 The samples of bronchial blockage sites of typical dead chickens in different chicken farms were collected aseptically. The bacteriological analysis was carried out by conventional bacterial isolation method combined with 1 6S DNA sequencing, and the drug sensitivity test and pathogenicity test were carried out. 【Results】 The results showed that E. coli and E.faecalis were isolated from 18 samples collected from 11 chicken farms, among which Proteus Mirabilis was isolated from 2 chicken farms and Acinetobacter Baumannii was isolated from 1 chicken farm. Mycoplasma Galliscepticum and Mycoplasma Synoviae were not detected in all the typical chicken farms. K-B drug susceptibility test showed that Escherichia coli were resistant to Ceftaxime, Chlorampenicol, Ampicillin, Tetracycline and Compound sulfamethoxazole. Enterococcus faecali were resistant to Ceftazidime, Aztreonam and Compound sulfamethoxazole. all isolates had no pathogenicity.【Conclusion】 This study shows that Escherichia coli, Enterococcus faecali, Acinetobacter baumannii and Proteus mirabilis can exist alone or together in the occluded Bronchi, which may be the main synergistic factor in the formation of this pathological change, while Escherichia coli and Enterococcus faecalis were found in all detected cases with strong resistance to antimicrobial drugs.

Key words: chicken bronchial obstruction; Escherichia coli; Enterococcus faecalis

中图分类号:

S831.7

刘璐瑶, 孙雪, 马万鹏, 王琦, 刘肖利, 苏战强. 鸡支气管堵塞死鸡病变部位感染细菌及其耐药性分析[J]. 新疆农业科学, 2021, 58(11): 2139-2147.

LIU Luyao, SUN Xue, MA Wanpeng, WANG Qi, LIU Xiaoli, SU Zhanqiang. Bacteriology and Drug Resistance Analysis of Dead Chickens with Bronchial Obstruction[J]. Xinjiang Agricultural Sciences, 2021, 58(11): 2139-2147.

图/表 12

参考文献 28

[1]	徐延青, 胡兆军: 肉鸡支气管阻塞的诊治[J]. 山东畜牧兽医, 2011, 32(2):62-63.
	XU Yanqing, HU Zhaojun. Diagnosis and treatment of bronchial obstruction in Broilers[J]. Shandong Animal Husbandry and Veterinary Medicine, 2011, 32(2):62-63.
[2]	王会娟, 闫冰广, 张瑞刚, 等. 一例蛋雏鸡因温度低引起的支气管堵塞的治疗[J]. 北方牧业, 2013, (8):24.
	WANG Huijuan, YAN Bingguan, ZHANG Ruigang, et al. Treatment of bronchial obstruction caused by low temperature in an egg chicken[J]. Northern Animal Husbandry, 2013,(8):24.
[3]	崔力兵, 叶晴, 耿士忠, 等. 治疗鸡支气管干酪样物质阻塞的两种临床新措施[J]. 今日畜牧兽医, 2010,(10):52.
	CUI Libing, YE Qing, GENG Shizhong, et al. Two new clinical measures for treating cheese-like substance obstruction in chicken bronchi[J]. Animal Husbandry and Veterinary Medicine Today, 2010,(10):52.
[4]	陈健: 一例商品蛋鸡气管堵塞的诊治[J]. 畜牧兽医科技信息, 2018,(1):105.
	CHEN Jian. Diagnosis and treatment of tracheal obstruction in commercial laying hens[J]. Information on Animal Husbandry and Veterinary Science and Technology, 2018,(1):105.
[5]	黄淑坚, 龚中贵, 陈秀玲, 等. 呼吸道病型H9亚型禽流感病毒的分离与鉴定[J]. 畜牧与兽医, 2007, 39(3):47-49.
	HUANG Shujian, GONG Zhonggui, CHEN Xiuling, et al. Isolation and identification of avian influenza virus subtype H9 of respiratory diseases[J]. Animal Husbandry and Veterinary, 2007, 39(3):47-49.
[6]	苏战强, 杜晓杰, 买买提·黑牙斯丁, 等. 鸡支气管栓塞的临诊辨证与治疗[J]. 畜禽业, 2012,(2):78.
	SU Zhanqiang, DU Xiaojie, Maimaiti Heiyastin, et al. Clinic Diagnosis and Treatment of Chicken Bronchial Embolism[J]. Livestock and Poultry Industry, 2012,(2):78.
[7]	何晓玲, 罗冠群, 尤永君, 等. 肉鸡支气管堵塞的病因分析及防治措施[J]. 中国家禽, 2013, 35(21):58.
	HE Xiaoling, LUO Guanqun, YOU Yongjun, et al. Etiology analysis and prevention measures of bronchial obstruction in broilers[J]. Chinese Poultry, 2013, 35(21):58.
[8]	陈起飞: 鸡呼吸道堵塞性疾病的综合防控[J]. 兽医导刊, 2017,(7):43-44.
	CHEN Qifei. Comprehensive Prevention and Control of Chicken Respiratory Obstruction[J]. Veterinary Guide, 2017, (7):43-44.
[9]	姬小薇: 肠出血性大肠杆菌EHECO157:H7的分子进化研究[D]. 重庆:第三军医大学, 2010.
	JI Xiaowei. Molecular Evolution of EHECO157:H7 of Enterohemorrhagic Escherichia coli[D]. Chongqing: Third Military Medical University, 2010.
[10]	黎满香, 林荣高, 薛立群, 等. 粪肠球菌PCR检测方法的建立[J]. 中国兽医科学, 2010,(12):1255-1258.
	LI Manxiang, LIN Ronggao, XUE Liqun, et al. Establishment of a PCR assay for Enterococcus faecalis[J]. Chinese Veterinary Science, 2010,(12):1255-1258.
[11]	Allen M S, Lamontagne M G . Identification of and Composition Containing Polyphosphate Accumulating Bacteria[J]. Unt Scholarly Works, 2013.
[12]	张磊, 李颖, 王慧, 等. 3种禽支原体多重PCR检测方法的建立及应用[J]. 中国畜牧兽医, 2011, 38(8):77-80.
	ZHANG Lei, LI Ying, WANG Hui, et al. establishment and application of multiple PCR for detection of mycoplasma avium in 3 species[J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2011, 38(8):77-80.
[13]	陆承平. 兽医卫生微生物学(第五版)[M]. 北京, 中国农业出版社, 2013.
	LU Chengping. Veterinary Health Microbiology (Fifth Edition) [M]. Beijing, China Agriculture Press, 2013.
[14]	Clinical and Laboratory Standards Institute. M31-A3, Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals (3rd Ed.) [M].USA, 2015.
[15]	刘松涛: 肉鸡支气管堵塞并发大肠杆菌病的防治措施[J]. 吉林畜牧兽医, 2011, 32(12):24-25.
	LIU Songtao. Prevention and treatment of bronchial obstruction complicated with colibacillosis in broilers[J]. Jilin Animal Husbandry and Veterinary Medicine, 2011, 32(12):24-25.
[16]	Nakamura, K., et al., Pathologic and Immunohistochemical Studies of Newcastle Disease (ND) in Broiler Chickens Vaccinated with ND: Severe Nonpurulent Encephalitis and Necrotizing Pancreatitis[J]. Veterinary Pathology, 2008, 45(6):928-933. DOI PMID
[17]	Pan Q, Liu A, Zhang F, et al. Co-infection of broilers with Ornithobacterium rhinotracheale and H9N2 avian influenza virus[J]. BMC Veterinary Research, 2012, 8(1):104. DOI URL
[18]	倪云龙: 大肠杆菌也是狠角色[J]. 生命世界, 2011,(9):62-66.
	NI Yunlong. Escherichia coli is also a strong role[J]. Life World, 2011,(9):62-66.
[19]	韩镌竹, 李欣南, 苏葳艺, 等. 两种粪肠球菌的分离纯化方法比较[J]. 现代畜牧兽医, 2015,(1):5-9.
	HAN Juanzhu, LI Xinnan, SU Weiyi, et al. Comparison of two isolation methods of Enterococcus faecalis[J]. Modern Animal Husbandry and Veterinary Medicine, 2015,(1):5-9.
[20]	田素飞, 褚云卓, 年华, 等. 2002至2011年奇异变形杆菌的临床分布及药物敏感性变迁[J]. 中华检验医学杂志, 2012, 35(12):1180-1182.
	TIAN Sufei, CHU Yunzhuo, NIAN Hua, et al. Clinical distribution and drug sensitivity changes of Proteus mirabilis from 2002 to 2011[J]. Chinese Journal of Laboratory Medicine, 2012, 35(12):1180-1182.
[21]	苏向宏: 临床样本鲍曼氏不动杆菌检测及药敏试验结果分析[J]. 中国医学工程, 2015,(8):166.
	SU Xianghong. Detection of Acinetobacter baumannii in clinical samples and analysis of drug susceptibility test results[J]. China Medical Engineering, 2015,(8):166.
[22]	高菘. 我国部分地区禽大肠杆菌病流行病学、免疫保护机理和与低致病禽流感病毒的致病协同[D]. 扬州:扬州大学, 2001.
	GAO Song. Epidemiology, immunoprotection mechanism and pathogenesis of low-pathogenic avian influenza virus in some areas of China[D]. Yangzhou: Yangzhou University, 2001.
[23]	王亚芳, 崔小七, 贾召锋, 等. 鸡大肠杆菌病的模型建立与中兽医辨证论治研究进展[J]. 中国畜牧兽医, 2014, 41(9):269-272.
	WANG Yafang, CUI Xiaoqi, JIA Shaofeng, et al. Research progress of Chicken colibacillosis model establishment and syndrome differentiation and treatment of Chinese veterinary medicine[J]. Chinese Animal Husbandry and Veterinary Medicine, 2014, 41(9):269-272.
[24]	桑卫洪: 肠球菌感染的临床分布及耐药特征[J]. 中国医药导刊, 2010, 12(2):217-218.
	SANG Weihong: Clinical distribution and drug resistance characteristics of enterococcal infection[J]. Chinese Journal of Medicine, 2010, 12(2):217-218.
[25]	曲志娜, 胡学远, 王娟, 等. 不同省区鸡源大肠杆菌耐药性调查与系统进化群分析[J]. 中国兽药杂志, 2014, 48(9):4-8.
	QÜ Zhina, HU Xueyuan, WANG Juan, et al. Investigation of drug resistance and phylogenetic analysis of E. coli isolated from chickens in different provinces[J]. Chinese Journal of Veterinary Medicine, 2014, 48(9):4-8.
[26]	Domig K.J, H.K. Mayer and W. Kneifel, Methods used for the isolation, enumeration, characterization and identification of Enterococcus spp. 2. Pheno- and genotypic criteria[J]. International Journal of Food Microbiology, 2004, 88(2-3):165-88. DOI URL
[27]	苏崴艺, 李欣南, 韩镌竹, 等. 某养殖场鸡源粪肠球菌分离鉴定及耐药性研究[J]. 现代畜牧兽医, 2014,(4):48-50.
	SU Weiyi, LI Xinnan, HAN Juanzhu, et al. Identification and Drug Resistance of Enterococcus faecalis from chicken in a farm[J]. Modern Animal Husbandry and Veterinary Medicine, 2014,(4):48-50.
[28]	商军, 顾欣, 张文刚, 等. 猪、鸡源分离粪肠球菌的核糖体分型及耐药性分析[J]. 中国抗生素杂志, 2017, 42(3):230-236.
	SHANG Jun, GU Xin, ZHANG Wengang, et al. Ribosomal typing and drug resistance analysis of Enterococcus faecalis isolated from pigs and chickens[J]. Chinese Journal of Antibiotics, 2017, 42(3):230-236.

引物名称 Primer name	引物序列 Primer sequences (5'→3')	目的片段 Product length/bp
大肠杆菌 Escherichia coli	F:5'-ATCC TGGC TCAG ATTG AACG CT-3' R:5'-TACA AGGC CCGG GAAC GTAT TCA-3'	1 439 bp^[9]
粪肠球菌 Enterococcus faecalis	F:5'-TGCC GCAT GGCA TAAG AG-3' R:5'-TGCA TCAC CTCG CGGT CTAG-3'	1 271 bp^[10]
16S rDNA	27F:AGAGTTTGATCCTGGCTCAG 1492R:CGGTTACCTTGTTACGACTTC	1 500 bp^[11]
鸡毒支原体MG Mycoplasma gallisepticum	F:5'-GGATCCCATCTCGACCAGGAGAAAA-3' R:5'-CTTTCAATCAGTGAGTAACTGATGA-3'	732 bp^[12]
滑液支原体MS Mycoplasma synoviae	F:5'-GAAGCAAAATAGTGATATCA-3' R:5'-GTCGTCTCGAAGTTAACAA-3'	207 bp^[12]

引物名称 Primer name	引物序列 Primer sequences (5'→3')	目的片段 Product length/bp
大肠杆菌 Escherichia coli	F:5'-ATCC TGGC TCAG ATTG AACG CT-3' R:5'-TACA AGGC CCGG GAAC GTAT TCA-3'	1 439 bp^[9]
粪肠球菌 Enterococcus faecalis	F:5'-TGCC GCAT GGCA TAAG AG-3' R:5'-TGCA TCAC CTCG CGGT CTAG-3'	1 271 bp^[10]
16S rDNA	27F:AGAGTTTGATCCTGGCTCAG 1492R:CGGTTACCTTGTTACGACTTC	1 500 bp^[11]
鸡毒支原体MG Mycoplasma gallisepticum	F:5'-GGATCCCATCTCGACCAGGAGAAAA-3' R:5'-CTTTCAATCAGTGAGTAACTGATGA-3'	732 bp^[12]
滑液支原体MS Mycoplasma synoviae	F:5'-GAAGCAAAATAGTGATATCA-3' R:5'-GTCGTCTCGAAGTTAACAA-3'	207 bp^[12]

菌株 Strains	科马嘉尿路显色培养基生长状况 Growth status of CHROMagar^TM	绵羊血平板生长状况 Growth status of sheep blood plate	镜检结果 Microscopic examination results
1类菌 Class 1 bacteria	紫红色,不透明,边缘整齐表面光滑。	灰白色,半透明,边缘整齐表面光滑,γ溶血,直径2~3 mm。	革兰氏阴性短杆菌,两端重染呈哑铃状
2类菌 Class 2 bacteria	深蓝色菌落,不透明,边缘整齐表面光滑。	灰白色,不透明,边缘整齐表面光滑,γ溶血,直径0.5~1 mm。	革兰氏染色不明显,镜下为小的椭圆形,个别呈短链状排列。
3类菌 Class 3 bacteria	白色菌落,不透明,体积较大,无扩散迁徙的现象。	灰白色,不透明,边缘不整齐,呈散射状迁徙生长,γ溶血。	革兰氏阴性杆菌
4类菌 Class 4 bacteria	奶油色菌落,不透明	灰白色,不透明,边缘整齐,表面光滑,γ溶血,直径1~2 mm。	革兰氏染色不明显,镜检短杆状或球状

菌株 Strains	科马嘉尿路显色培养基生长状况 Growth status of CHROMagar^TM	绵羊血平板生长状况 Growth status of sheep blood plate	镜检结果 Microscopic examination results
1类菌 Class 1 bacteria	紫红色,不透明,边缘整齐表面光滑。	灰白色,半透明,边缘整齐表面光滑,γ溶血,直径2~3 mm。	革兰氏阴性短杆菌,两端重染呈哑铃状
2类菌 Class 2 bacteria	深蓝色菌落,不透明,边缘整齐表面光滑。	灰白色,不透明,边缘整齐表面光滑,γ溶血,直径0.5~1 mm。	革兰氏染色不明显,镜下为小的椭圆形,个别呈短链状排列。
3类菌 Class 3 bacteria	白色菌落,不透明,体积较大,无扩散迁徙的现象。	灰白色,不透明,边缘不整齐,呈散射状迁徙生长,γ溶血。	革兰氏阴性杆菌
4类菌 Class 4 bacteria	奶油色菌落,不透明	灰白色,不透明,边缘整齐,表面光滑,γ溶血,直径1~2 mm。	革兰氏染色不明显,镜检短杆状或球状

抗菌药物 Antibacterials	大肠杆菌耐药(R)(n=18) Drug resistance of Escherichia coil(R)(n=18)		粪肠球菌耐药(R)(n=18) Drug resistance of Enterococcus faecalis(n=18)
抗菌药物 Antibacterials	株数 Number of trees	耐药率 Drug resistance rate(%)	株数 Number of trees	耐药率 Drug resistance rate(%)
CTX	17	94.4	15	83.3
CAZ	10	55.5	16	89.8
AN	5	28.7	11	61.1
AZT	7	39.8	18	100
SMZ	18	100	16	89.8
CN	6	33.3	5	28.7
TE	18	100	11	61.1
Amp	18	100	2	11.1
CIP	13	72.2	6	33.3
LEV	9	50.0	6	33.3
CHL	14	78.7	9	50.0